Regulation of the Cardiac Na/Ca Exchanger

描述(由申请人提供)：心脏钠/钙交换器(NCX)是心肌细胞中主要的钙外流机制，在调节心肌收缩能力方面发挥着重要作用。表征交换器的调节特性对于理解正常的心脏生理和在致病条件下发生的扰动都很重要。本项目主要研究细胞内钙离子对NCX活性的变构调节以及细胞骨架对其调节作用。指导这些研究的假设是，变构钙激活表现出滞后，因此在NCX激活(持续性钙激活)后，活性变得不依赖于钙。我们认为，这种滞后是由于(A)涉及NCX蛋白“β重复区域”的细胞骨架相互作用，限制了钙对调节结合部位的访问，以及(B)PIP2与NCX蛋白所谓的XIP区域的相互作用。该项目针对以下目标有5个具体目标：(1)胞浆[Na]升高诱导NCX活性的钙非依赖性模式。我们将测量这一反应的[Na]依赖性，并研究细胞骨架相互作用、PIP2和XIP突变在这一活动发展中的作用。(2)我们将确定蛋白激酶C、NO供体或激活的RAC或CdC42对F-肌动蛋白动力学的刺激是否促进了钙非依赖性活动的发展，并加快了变构钙激活的速度。(3)我们将确定细胞骨架相互作用、胞内[Na]和PIP2如何影响持续性钙激活的发展和松弛。(4)将在交换器的β重复区域产生突变，并评估它们对细胞骨架相互作用、变构钙激活和钙非依赖性NCX活性的影响。(5)我们将确定细胞骨架相互作用是否调节新生和成年大鼠心肌细胞中NCX的活性。转基因的CHO细胞将用于AIMS 1-4。使用荧光钙探针的数字成像技术将被用来测量NCX的运输活性。

DESCRIPTION (provided by applicant): The cardiac Na/Ca exchanger (NCX) is the principal Ca efflux mechanism in cardiac myocytes and plays an essential role in regulating cardiac contractility. Characterizing the exchanger's regulatory properties is important for understanding both normal cardiac physiology and the perturbations that occur in pathogenic conditions. This project focuses on the allosteric regulation of NCX activity by cytosolic Ca and its modulation by the cytoskeleton. The hypothesis guiding these studies is that allosteric Ca activation displays hysteresis so that activity becomes Ca-independent following NCX activation (persistent Ca activation). We propose that the hysteresis is due to (a) cytoskeletal interactions involving the "beta repeat regions" of the NCX protein that restrict access of Ca to the regulatory binding sites and (b) interaction of PIP2 with the so- called XIP region of the NCX protein. The project has 5 specific aims directed toward the following goals: (1) Elevations in cytosolic [Na] induce a Ca-independent mode of NCX activity. We will measure the [Na] dependence of this response and investigate the role of cytoskeletal interactions, PIP2 and XIP mutations in the development of this activity. (2) We will determine whether stimulation of F-actin dynamics by protein kinase C, NO donors or activated Rac or Cdc42 enhances the development of Ca-independent activity and accelerates the rate of allosteric Ca activation. (3) We will determine how cytoskeletal interactions, cytosolic [Na] and PIP2 influence the development and relaxation of persistent Ca activation. (4) Mutations will be created in the beta repeat regions of the exchanger and their influence on cytoskeletal interactions, allosteric Ca activation and Ca-independent NCX activity will be assessed. (5) We will determine whether cytoskeletal interactions modulate NCX activity In rat neonatal and adult cardiac myocytes. Transfected CHO cells will be used in Aims 1-4. Digital imaging techniques using fluorescent Ca probes will be used to measure NCX transport activity.